In vitro propagation of grape

ABSTRACT

A process for controlling the development of somatic embryos for either a self-replicative or a vine development mode. Somatic embryogenesis is initiated by placing the embryo in a medium conducive for self-replication. When the embryos have grown to a first stage, they are transferred to a medium with cytokinin activity. They continue to grow until they have grown to a second stage at which time they are transferred to a medium without cytokinin activity until they have grown sufficiently and can be transferred to a non-liquid growth medium.

The present invention embodies a method of clonal propagation of certainangiosperms and particularly, the hybrid cultivar "Seyval" of the genusVitis (grapes).

BACKGROUND AND BRIEF SUMMARY OF THE INVENTION

Prior art concerning the propagation of the Vitis species by tissueculture methods is not that prevalent. Currently, there is one reportwhich deals with vine producton from fragmented shoot apices; M. Barlassand K. G. M. Skeen (1978), "In vitro propagation of grapevine (Vitisvinifera L.) from fragmented shoot apices." Vitis 17:335-340. There aretwo reports which demonstrate the feasibility of producing vines viasomatic embryogenesis, Krul and Worley, "Formation of AdventitiousEmbryo in Callus Cultures of `Seyval`", J. Amer. Soc. Hort. Sci.102:360-363; and M. G. Mullins et al (1976), Somatic Embryos andPlantlets from an Ancient Clone of the Grapevine (cv.Cabernet-Sauvignon) by apomixis in vitro. J. Expt. Bot. 27:1022-1030. Itis possitle that grape propagation by shoot tip multiplication mayrequire little or no modification of established methods and therefore,is not reported. See T. Murashige (1974), Plant Propagation ThroughTissue Cultures, Annu. Rev. Plant Physiol. 25:135-166.

In plant propagation, by somatic embryogenesis, i.e. the production ofembryonic structures (identical to those in seeds) from isolated plantcells was described in the late 1950's by F. C. Steward et al (1958),Growth and Organized Development of Cultured Cells; II. Organization incultures grown from freely suspended cells. Amer. J. Bot. 45:705-708;and J. Reinert, Uber die Kontrolle der orphogenese und die Induktion vonAdventivembryonen an Gewebekulturen aus Karotten, Planta 53:318-333.

Embryoids of grape such as other plants arise from proembryonic massesembedded in callus cells. The factors involved in the initiation ofproembryonic masses are not known. The transformation of theproembryonic masses to embryoids in grape occurs when they are shiftedfrom a medium containing a active auxin to one containing a less activeone; or when the cytokinin level of the medium is lowered. A highlyembryogenic callus of `Seyval` has been isolated which does not requiregrowth regulators for growth. Embryoid formation in this callus occurswhen it is grown on auxin for a short while and then cultured onauxin-free media, when the calcium concentration is one half that of theMurashige-Skoog formulation, or when the cultures are grown for longintervals without subculture. Most of the vines regenerated from theoriginal `Seyval` callus have appeared to be normal. However, asubpopulation of these embryos did not develop normally but producedsecondary somatic grape embryos at the junction of the root and shoot.

The development of normal vines from these etiolated secondary grapeembryoids is complicated by separate dormancies of the roots, hypocotol,cotyledons and shoot apices, each of which respond to different physicaland chemical stimuli.

The processes of successful embryoid production and normal vinedevelopment have appeared to be mutually antagonistic. For instance,embryoids which produce embryoids generally senesce and die shortlyafter embryoid formation. In contrast, embryoids which develop flattenedgreen separate cotyledons or normal shoots generally lose their capacityfor the production of secondary embryoids. Therefore, treatments whichencourage embryoid production do not favor normal vine development, andconversely, treatments which favor normal vine development restrictsomatic embryogenesis.

The production of vines or other plants from isolated cells via somaticembryogenesis is essential for progress in anticipated geneticmodifications for plant improvement. Such strategies may involvemanipulation in selections of cells tolerant to physical stress (cold orheat) or chemical stress, (herbicidal, bacterial and fungal toxins) orthe uptake of gene vectors by protoplasts from embryogenic cells. Theuse of somatic embryogenesis as an alternative method for the massiveproduction of plants in particular vines has possibilities, butheretofore methods for most cultivars have yet to be developed.

The basic procedures for the mass clonal propagation of certain plantsvia epidermal cells of somatic embryos is set forth in a publication "InVitro Propagation of Grape", W. R. Krul and Myerson (1980); JournalArticle Number 1932 Rhode Island Agricultural Experimental Station.Also, a detailed discussion of this field is found in a thesis entitled"Adventitious Embryo-genesis and Plantlet Development in Cultures ofVitis vinifera L. `Seyval`", Judith Myerson thesis submitted to theUniversity of Rhode Island presently on file at the Library of theUniversity of Rhode Island, Kingston, R.I.

I have discovered a method and the plants produced thereby in whichsomatic embryos may assume either a self-replicating mode or a vinedevelopment mode. Prior to my present invention, when a process wasemployed which was successful in producing self-replicating embryos, thedevelopment of normal plants from such embryos occurred at a frequencyof about 5%. The present invention permits development of functionalplants from such embryos at a frequency in excess of 90%. With thepresent invention, it is possible to maintain a number of plants in thereplicative mode and induce functional plants at times convenient for aproducton schedule. The invention is exemplified through the clonalpropagation of grape via epidermal cells of somatic embryos of the Vitiscultivar specifically the hybrid `Seyval`.

Broadly, the cloning process includes removing somatic embryos from a`mother` plant (see the Drawing, cycle A). Such embryos are cultured fora first time period on a medium, (M/S medium without growth regulators).In this medium, the embryos self-replicate. Subsequently, those embryoswhich are selected for vine development (see the Drawing, cycle B) aretransferred to the same medium with a growth regulator having cytokininactivity and maintained for a fixed period of time in the medium untilthe desired level of embryo development is reached. At this stage, theembryos are transferred to the same medium without the growth regulatorand allowed to develop to allow transplant to soil.

BRIEF DESCRIPTION OF THE DRAWING

The drawing is a schematic of the life cycle of a secondary somaticembryo.

DESCRIPTION OF THE PREFERRED EMBODIMENT

The preferred embodiment of the invention will be described in referenceto embryo development in self-replicative and vine development modes.

The maintenance of mother plants and removal of embryos from a motherplant is described in the above-referenced thesis, "AdventitiousEmbryogenesis and Plantlet Development in Cultures of Vitis viniferaL.", `Seyval`, which thesis is hereby incorported by reference in itsentirety in this disclosure.

Adventitious embryogenesis, that is, the initiation of embryos directlyfrom the somatic cells of another embryo or plantlet in culture is notunusual. In the specific non-limiting example set forth below,adventitious embryos, formed on the surface of the root/shoot transitionzone of developing `Seyval`plantlets regenerated from callus, werecultured. These embryos initiated other embryos that also producedembryos, i.e. cloning. Typically, a mother plant will contain anywherefrom 0 to 100 such embryos. As is known, it is not usually necessary tophysically detach adventitious embryos from the transition zone of theparent plant since most embryos germinate and separate from the parentplant spontaneously.

The life cycle of a secondary somatic embryo of the Vitis species`Seyval` is illustrated in the FIGURE. Cycle A, the self replicatingmode, illustrates the progress of a torpedo stage embryo, through itsexpansion stage and finally its "reproductive" stage. The cycle requires30-40 days for completion. Such embryos, when grown on Murashige andSkoog (1962) mineral salts and vitamins supplemented with sucrose andwith no plant growth regulators, will remain in the "reproductive" mode,indefinitely. To obtain normal vine development, (pathway B) the torpedostage embryo, is transferred to M+S media (as above), supplemented with2.5 uM benzyladenine (BA) for a period of 7 days. When cotyledons becomegreen and begin to separate, the embryo is removed from this medium andplaced on the same medium (M+S without BA) until ready for transplantingto a soil mix (approximately 30 days).

Although specific example will be described with reference to `Seyval`wherein adventitious embryogenesis occurs spontaneously from the roottransition zone of plant embryos, without an intervening callus phase,the invention also contemplates embryogenesis or plant development bythe following methods of embryo initiation, for example, other types ofsomatic embryogenesis within the scope of the invention are from callus,from anther or pollen cultures, tumor cells or those transformed by theaddition of foreign genes.

Somatic embryos of Vitis sp. `Seyval` produce secondary somatic embryosfrom epidermal cells at the transition zone between the root and shoot.This process of embryo multiplication (pathway A) occurs when embryosare cultured on MS media plus organics and 3% sucrose (hormone-freemedia). By transferring embryos produced on mother embryos tohormone-free media, secondary somatic embryogenesis proceedsindefinitely. Most (95%) of the embryos produced do not develop intonormal vines; but instead, are arrested at a developmental stage whichfavors the production of secondary embryos rather than normal shootdevelopment (pathway A, The FIGURE). In the embryo multiplication phase,the embryos have characteristically white unexpanded wrinkled cotyledons(instead of smooth and green) and a dormant shoot apex. About 5% ofthese embryos develop into normal vines through a process which firstinvolves greening of the cotyledons, and next the growth of the shootapex (pathway B). It would be desirable to have a means to enhancenormal vine development so plants could be multiplied utilizing theembryo multiplication pathway (A) and normal vines obtained bysubjecting the somatic embryos to a specific treatment.

The results of the following experiment (EXAMPLE) teaches a processwhich will enhance normal vine development from somatic embryos (pathwayB). The experiment investigated the effects of benzyl adenine on thedevelopment of somatic embryos into normal vines (pathway B). Somaticembryo of three different size classes were used to determine if embryosize affects susceptibility to benzyl adenine treatment. Small embryoswere of late torpedo stage are characterized by their size (2mm), theircolor (white), and the absence of an expanding root or shoot apex.Medium size embryos (2-6mm) are characterized by their small primaryroot, small green stem, and white, fused, wrinkled cotyledons. Largeembryos are morphologically similar to medium-size embryos but arelarger (6-10 mm).

Somatic embryos (cycle A) from the root-hypocotyl transition zone ofmother embryos were transferred to either MS media plus vitamins, 3%sucrose, and 2.5 μM benzyl adenine (BA media) or MS media plus vitaminsand 3% sucrose (hormone-free media). After one week of growth on BAmedia or hormone-free media, embryos were transferred to hormone-freemedia. During this week, cotyledons of embryos grown on BA greened andshoot development began. However, the cotyledons of untreated embryosremained white and the shoot apex dormant (Table I). BA treatment wasmost effective on small embryos (Table I). Once BA treated embryos weretransferred to hormone-free media, normal shoot development proceededand normal vines were obtained without further BA treatment. If largerembryos were used for the BA treatment, fewer normal vines were obtained(Tables I, II, and III). If embryos were grown on BA media for longerthan one week, the cotyledons grew abnormally large and leafy and normalvines were not obtained (data not shown). Embryos not treated with BAundergo secondary somatic embryogenesis and produce as many as 70embryos per mother embryo after 38 days. Embryos treated with BA for oneweek also produce secondary embryos but generally not to the same extentas untreated embryos.

EXAMPLE

Embryos were removed from mother plants and cultured in hormone-freemedia (cycle A) and removed when they reach the reproductive stage after30 to 40 days. The removed embryos were classified according to small,medium and large; small (late torpedo stage) consisting of embryos lessthan 2 mm; medium (beginning to germinate--root and shoot) consisting ofembryos 2-6 mm; and large (older embryos with root and white dividedcotyledon) consisting of embryos of 6-10 mm. Sixty embryos, 20 small, 20medium and 20 large, were maintained for seven (7) days 10 small, 10medium and 10 large embryos were maintained in hormone-free media; and10 small, 10 medium and 10 large embroys were maintained in BA media in25×150 mm tubes (sealed with kaputs). The BA media comprised 10 ml ofMurashige and Skoog media, including vitamin supplements, with 3%sucrose, 0.8% agar, 2.5 μM BA and pH adjusted to 6 with KOH; (stockculture same as culture of mother plant). The hormone-free media wasidentical less the BA. The final media pH after autoclaving was 5.7. Thetubes were placed on 45 ° slant racks under fluoroscent lights (10×25uE^(m-2s-1)), 16 hours photoperiod followed by eight hours darkness, atambient temperatures of between 21°-27° C.

During the seven (7) days, the embryos began to germinate, that is,develop minute shoots, roots, etc. Table I below sets forth thecharacteristics of the embryos after one week culture in hormone-freemedia and BA media.

Subsequently, the 10 small, 10 medium and 10 large embryos maintained inthe hormone-free media were allowed to continue to culture inhormone-free media for an additional seven (7) days, while the 10 small,10 medium and 10 large embryos maintained in the BA media weretransferred to a hormone-free media as described above. Table II setsforth the plant development of those embryos maintained in the mediumwith cytokinin activity compared with those embryos maintained in mediumwithout cytokinin activity.

                                      TABLE I                                     __________________________________________________________________________    The effect of BA on development of semi-dormant                               secondary embryos after one week of treatment.                                Incubator: 28° C.  16 hr light; 8 hr dark                              Treatment                                                                           Size of                                                                 Media Initial Embryo                                                                        1   2   3   4   5   6   7   8   9   10  %   %                   __________________________________________________________________________                  PD  #2° embryos                                          BA    Small   GC-0                                                                              GC-0                                                                              GC-0                                                                              GC-0                                                                              WC-0                                                                              GC-0                                                                              GC-0                                                                              GC-0                                                                              GC-0                                                                              GC-0                                                                              90                                                                                10 WC               HF    Small   WC-0                                                                              WC-0                                                                              WC-0                                                                              WC-0                                                                              WC-0                                                                              WC-0                                                                              WC-0                                                                              WC-0                                                                              WC-0                                                                              WC-0    100 WC              BA    Medium  GC-0                                                                              WC-0                                                                              WC-0                                                                              WC-0                                                                              WC-0                                                                              GC-0                                                                              WC-0                                                                              WC-0                                                                              GC-0                                                                              GC-0                                                                              40                                                                                60 WC               HF    Medium  WC-0                                                                              WC-0                                                                              WC-0                                                                              WC-0                                                                              WC-0                                                                              WC-0                                                                              WC-2                                                                              WC-0                                                                              WC-0                                                                              WC-0    100 WC              BA    Large   WC-0                                                                              GC-0                                                                              WC-0                                                                              WC-0                                                                              GC-0                                                                              WC-0                                                                              WC-0                                                                              WC-0                                                                              WC-0                                                                              WC-0                                                                              20                                                                                80 WC               HF    Large   WC-0                                                                              WC-0                                                                              WC-0                                                                              WC-0                                                                              WC-0                                                                              WC-0                                                                              WC-1                                                                              WC-2                                                                              WC-0                                                                              WC-0    100                 __________________________________________________________________________                                                              WC                   Treatment Media                                                                BA = Benzyl Adenine                                                          HF = Hormone Free                                                             Plant Development (PD)                                                        GC = Green Cotyledon                                                          WC = White Cotyledon                                                     

                                      TABLE II                                    __________________________________________________________________________    The effect of BA treatment on development of                                  semi-dormant somatic embryos after one week                                   of treatment and one week of growth hormone                                   free media.                                                                   __________________________________________________________________________    Treatment                                                                           Embryo Size Prior                                                       Media to Treatment                                                                            1   2    3   4   5   6   7                                    __________________________________________________________________________                    PD  #2° Embryos                                        BA    Small     GCA-0                                                                             GC-0 GC-1                                                                              GC-0                                                                              WC-0                                                                              GCA-0                                                                             GCA-0                                HF    Small     WC-0                                                                              WC-0 WC-0                                                                              WC-0                                                                              WC-0                                                                              WC-0                                                                              WC-0                                 BA    Medium    GCA-5                                                                             WCA-6                                                                              WC-0                                                                              WC-0                                                                              WC-5                                                                              GC-1                                                                              WC-0                                 HF    Medium    WC-0                                                                              WC-0 WC-0                                                                              WC-0                                                                              WC-0                                                                              WC-3                                                                              WC-1                                 BA    Large     WC-0                                                                              GC-0 WC-2                                                                              WC-0                                                                              GC-0                                                                              WC-0                                                                              WC-0                                 HF    Large     WC-0                                                                              WC-0 WC-0                                                                              WC-1                                                                              WC-0                                                                              WC-3                                                                              WC-1                                 __________________________________________________________________________    Treatment                                                                           Embryo Size Prior                                                       Media to Treatment                                                                            8   9   10  NUMBER                                            __________________________________________________________________________    BA    Small     P-0 GC-1                                                                              GC-0                                                                              1 P  3 GCA                                                                             5 GC                                                                              1 WC                                 HF    Small     WC-0                                                                              WC-2                                                                              WC-0             10 WC                                BA    Medium    WC-0                                                                              GC-0                                                                              GC-0                                                                              1 WCA                                                                              1 GCA                                                                             3 GC                                                                              5 WC                                 HF    Medium    WC-2                                                                              WC-1                                                                              WC-2             10 WC                                BA    Large     WC-1                                                                              WC-3                                                                              WC-0                                                  HF    Large     WC-2                                                                              WC-0                                                                              WC-0                                                                              2 GC 8 WC    10 WC                                __________________________________________________________________________     Treatment Media                                                               BA = Benzyl Adenine                                                           HF = Hormone Free                                                             Plant Development (PD)                                                        WC = White Cotyledon                                                          GC = Green Cotyledon                                                          A = Apex between GC or WC                                                     P = Plant                                                                

                  TABLE III                                                       ______________________________________                                        The effect of BA treatment on normal plant development                        after one week of treatment and four weeks growth on                          hormone free media.                                                                                 % Normal Plants                                                               (after one week treatment                               Treatment                                                                             Embryo Size (mm)                                                                            and three weeks growth                                  Media   Before Treatment                                                                            on hormone free media)                                  ______________________________________                                        BA      Small (<2 mm) 90                                                      HF      Small         0                                                       BA      Medium (2-6 mm)                                                                             40                                                      HF      Medium        0                                                       BA      Large (6-10 mm)                                                                             20                                                      HF      Large         0                                                       ______________________________________                                    

The embryos maintained in the media containing the cytokinin wereallowed to continue to culture in cytokinin-free media as describedabove. They ultimately developed into normal plants. This is set forthin Table III.

Other cytokinins, both natural and synthetic, may be used to permit thetransition from the embryo replicative mode to vine development mode.That is, with the present invention, if it is desired to keep the plantin the self-replicative mode, it is not transferred to the medium withthe cytokinin activity, but rather is allowed to continue to develop,which development will result in poor to nonexistent plant development,but very satisfactory embryo multiplication. Where it is desired to havethe embryos enter the vine development mode, then they are transferredto the medium with the cytokinin activity as just described.

The invention has been described with reference to the cytokininactivity of 2.5 μM benzyl adenine. Within the scope of the invention,other natural and synthetic cytokinins which will permit transition fromthe embryo replicative mode to the normal vine growth and developmentmode include zeatin and its riboside and ribotide; isopentenyl adenineand its riboside and ribotide; kinetin; ethyoxyethyladenine; 2-2hydroxyzeatin; N,N¹ -diphenylurea and 8-azakinetin. Although describedin reference to a concentration of cytokinin activity of 2.5 μM, withinthe scope of the invention various concentrations of cytokinin whichwill promote the desired activity are contemplated. The cytokinins maybe used alone or in combination.

It is believed that two factors play an important part in thedevelopment of normal plants from these embryos, namely the size of theembryo when transferred to the medium with cytokinin activity and thelength of time that the plant is allowed to remain in the medium withthe cytokinin activity. More specifically, control of the growth of theembryos in any suitable medium until such time as the embryos evidencesigns of germinating, i.e. development of roots, shoots, etc., is thefirst step. When the embryo should physically be removed for transfer tomedia with cytokinin activity, the embryo may be generally characterizedby the following: (a) less than 2mm long, consisting of visiblecotyledon, hypocotyl and root. The color of the embryo is mostly anopaque ivory white with the root zone characterized by yellowishtranslucent tissue. The cotyledons are appressed and have not yetcompleted expansion and final shape is not yet assumed. Approximately1/3 of the embryo mass consist of cotyledonary tissue. The root zoneappears to be in the primorida stage, e.g. still in the cell divisionstage, as no cell elongation is obvious. The root mass is approximately1/4 (or less) of the total embryo. The correct morphological descriptorfor embryo at this stage of development is late torpedo stage.

After embryo development has reached the stage as described above, if itis desired that the embryos should continue to development toself-replicative (i.e. produce more embryos), then they are simplyallowed to remain in the same or a similar medium. If it is desired thatthe embryos be transferred to the vine development mode, then they aretransferred to a medium with cytokinin activity. The duration of timeduring which the embryos remain in the medium with the cytokininactivity for a fixed period of time is critical. This fixed period oftime is sufficient to insure that the embryos as germinating do notbecome either underdeveloped or overdeveloped, in which cases normalvine development will not occur. The development stage at which theembryos must be removed may be described as follows: cotyledons dividedand green--apex may or may not be visible.

Although described in reference to the hybrid `Seyval`, other plantsclassified as follows are within the scope of my invention: All generawith the potential to form somatic embryo from isolated cells.

Having described my invention, what I now claim is:
 1. A method forcontrolling the growth of plant embryos for either self-replicative orvine development modes, which includes:(a) initiating somaticembryogenesis by placing somatic embryos in a medium conducive forself-replication of the embryos for a first period of time during whichthe embryos germinate and are characterized by late torpedo stageembryos prior to root and shoot elongation; (b) culturing for vinedevelopment at least some of the embryos of step (a) in a medium havingcytokinin activity for a second period of time at the end of which timethe embryos are characterized by green divided cotyledons; (c)transferring the embryo of step (b) to a medium free of cytokininactivity until such time that they develop sufficiently for transfer toa non-agar growth support medium.
 2. The method of claim 1 wherein theplant embryos are selected from plants which are capable of formingsomatic embryos from isolated cells of the plant.
 3. The method of claim1 wherein the embryos initiated are selected from the group consistingof:callus, anther or pollen cultures and/or tumor cells.
 4. The methodof claim 1 wherein the cytokinin activity of the medium of step (b) iseffected by the addition of an effective amount of a cytokinin selectedfrom the group consisting of zeatin and its riboside and ribotide,isopentenyl adenine and its riboside and ribotide; kinetin,ethyoxyethyladenine; 2-2 hydroxyzeatin; N,N diphenylurea and benzyladenine and/or 8-azakinetin.
 5. The method of claim 4 wherein thecytokinin is benzyl adenine.